Product Manual

OxiSelect™ ROS Assay Kit

Catalog Number

STA-342 96 assays

FOR RESEARCH USE ONLY Not for use in diagnostic procedures

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Introduction Accumulation of reactive oxygen species (ROS) coupled with an increase in oxidative stress has been implicated in the pathogenesis of several disease states. The role of oxidative stress in vascular diseases, diabetes, renal ischemia, atherosclerosis, pulmonary pathological states, inflammatory diseases, and cancer has been well established. Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check by the existence of multiple antioxidant and repair systems as well as the replacement of damaged nucleic acids, proteins and lipids. Measuring the effect of antioxidant therapies and ROS activity intracellularly is crucial to suppressing or treating oxidative stress inducers. Cell Biolabs’ OxiSelect™ ROS Assay Kit is a cell-based assay for measuring hydroxyl, peroxyl, or other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe 2’, 7’-Dichlorodihydrofluorescin diacetate (DCFH-DA). In brief, DCFH-DA is diffused into cells and is deacetylated by cellular esterases to non-fluorescent 2’, 7’-Dichlorodihydrofluorescin (DCFH), which is rapidly oxidized to highly fluorescent 2’, 7’-Dichlorodihydrofluorescein (DCF) by ROS (Figure 1). The fluorescence intensity is proportional to the ROS levels within the cell cytosol. The effect of antioxidant or free radical compounds on DCF-DA can be measured against the fluorescence of the provided DCF standard. The kit has a DCF detection sensitivity limit of 10 pM. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples. Assay Principle The OxiSelect™ ROS Assay Kit is a cell-based assay for measuring antioxidant or ROS activity. Cells are cultured in a 96-well cell culture plate and then pre-incubated with DCFH-DA, which is cell-permeable. The unknown antioxidant or ROS samples are then added to the cells. After a brief incubation, the cells can be read on a standard fluorescence plate reader. The ROS or antioxidant content in unknown samples is determined by comparison with the predetermined DCF standard curve. Related Products

1. STA-305: OxiSelect™ Nitrotyrosine ELISA Kit

2. STA-310: OxiSelect™ Protein Carbonyl ELISA Kit

3. STA-330: OxiSelect™ TBARS Assay Kit (MDA Quantitation)

4. STA-332: OxiSelect™ MDA ELISA Kit

5. STA-334: OxiSelect™ HNE Adduct ELISA Kit

6. STA-340: OxiSelect™ Superoxide Dismutase Activity Assay

7. STA-341: OxiSelect™ Catalase Activity Assay Kit

8. STA-345: OxiSelect™ ORAC Activity Assay

9. STA-346: OxiSelect™ HORAC Activity Assay

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Figure 1. Mechanism of DCF Assay Kit Components 1. 20X DCFH-DA (Part No. 234201): One 500 µL amber tube of a 20 mM solution in methanol.

2. DCF Standard (Part No. 234202): One 100 µL amber tube of a 1 mM solution in DMSO.

3. Hydrogen Peroxide (Part No. 234102): One 100 µL amber tube of an 8.821 M solution.

4. 2X Cell Lysis Buffer (Part No. 234203): One 20 mL bottle.

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Materials Not Supplied 1. Sterile DPBS for washes and buffer dilutions

2. Hank’s Balanced Salt Solution (HBSS)

3. Cell culture medium (ie: DMEM +/-10% FBS)

4. 37ºC Incubator, 5% CO2 atmosphere 5. 10 µL to 1000 µL adjustable single channel micropipettes with disposable tips 6. 50 µL to 300 µL adjustable multichannel micropipette with disposable tips 7. Multichannel micropipette reservoir 8. 96-well black or fluorescence microtiter plate 9. Cell culture microtiter plate(s) 10. Fluorescent microplate reader capable of reading 450 nm (excitation) and 530 nm (emission) Storage Upon receipt, store the DCFH-DA, DCF and Hydrogen Peroxide at -20ºC. Avoid multiple freeze/thaw cycles. Store the Cell Lysis Buffer at 4ºC. Preparation of Reagents • 1X DCFH-DA: Dilute the 20X DCFH-DA stock solution to 1X in cell culture media, preferably

without FBS. Stir or vortex to homogeneity. Prepare only enough for immediate applications.

• Hydrogen Peroxide (H2O2): Prepare H2O2 dilutions in DMEM or DPBS as necessary. Do not store diluted solutions.

Preparation of Standard Curve 1. Prepare a 1:10 dilution series of DCF standards in the concentration range of 0 µM – 10 µM by

diluting the 1mM DCF stock in cell culture media (see Table 1).

Table 1. Preparation of DCF Standards

Standard Tubes

DCF Standard (µL)

Culture Medium (µL)

DCF (nM)

1 10 990 10,000 2 100 of Tube #1 900 1000 3 100 of Tube #2 900 100 4 100 of Tube #3 900 10 5 100 of Tube #4 900 1 6 100 of Tube #5 900 0.1 7 100 of Tube #6 900 0.01 8 100 of Tube #7 900 0

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2. Transfer 100 µL of the DCF standard to a 96-well plate suitable for fluorescence measurement. Add 100 µL of the 2X Cell Lysis Buffer.

3. Read the fluorescence with a fluorescence plate reader at 480 nm excitation /530 nm emission.

Assay Protocol I. DCF Dye Loading

1. Prepare and mix all reagents thoroughly before use. Each cell sample, including unknown and standard, should be assayed in duplicate or triplicate.

2. Culture cells in either a clear or black 96-well cell culture plate. Note: If using a black plate, choose an appropriate plate based on your fluorometer’s reader

(i.e. choose a clear bottom black plate for bottom readers). 3. Remove media from all wells and discard. Wash cells gently with DPBS or HBSS 2-3 times.

Remove the last wash and discard. 4. Add 100 µL of 1X DCFH-DA/media solution to cells. Incubate at 37ºC for 30-60 minutes. 5. Remove solution. Repeat step three using multiple washes with DPBS or HBSS. Remove the

last wash and discard. 6. Treat DCFH-DA loaded cells with desired oxidant or antioxidant in 100 µL medium.

II. Quantitation of Fluorescence • Fluorescence microscopy or Flow cytometry: Fluorescence can be analyzed on an inverted

fluorescence microscope or by flow cytometry using excitation and emission wavelengths of 480 nm and 530 nm, respectively.

• Fluorescence Plate Reader: • Assays performed in black cell culture fluorometric plates: Plate may be read

immediately for kinetic analysis or after 1 hour for static analysis. Plates read for kinetic analysis may be read in increments of 1 and 5 minutes up to 1 hour or more as necessary. Read the fluorescence with a fluorometric plate reader at 480 nm/530 nm. Terminate the assay by adding 100 µL of the Cell Lysis Buffer to both standards and cell samples. Mix thoroughly and incubate 5 minutes. Read plate for final values.

• Assays performed in clear cell culture plates: Terminate the assay by adding 100 µL of the 2X Cell Lysis Buffer. Mix thoroughly and incubate 5 minutes. Transfer 150 µL of the mixture to a 96-well plate suitable for fluorescence measurement. Read the fluorescence with a fluorometric plate reader at 480 nm/530 nm.

Example of Results The following figures demonstrate typical ROS Assay results. Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer (Molecular Devices) with a 485/538 nm filter set and 530 nm cutoff. One should use the data below for reference only. This data should not be used to interpret actual results.

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0.1

1

10

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10000

100000

0.01 0.1 1 10 100 1000 10000

DCF (nM)

RFU

Figure 2. DCF Standard Curve.

0

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2000

0 µM 100 µM 1000 µM

H2O2 Concentration

RFU

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Figure 3. ROS in HeLa cells treated with H2O2. 50,000 HeLa cells in 96-well plate were first pretreated with 1 mM DCFH-DA for 60 minutes at 37ºC. Cells were then treated with H2O2 for 20 minutes.

Figure 4. DCF Fluorescence in H2O2 treated HeLa cells after 1 hour. Right: 0 µM; Left: 1000 µM. References

1. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J Immunol. 1983; 130:1910-1917.

2. Brandt R, Keston AS. Synthesis of diacetyldichlorofluorescin: A stable reagent for fluorometric analysis. Anal Biochem. 1965; 11:6-9.

3. Keston AS, Brandt R. The fluorometric analysis of ultramicro quantities of hydrogen peroxide. Anal Biochem.1965; 11:1-5.

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